Physiological tolerance and stoichiometric potential of cyanobacteria for hydrocarbon fuel production

Kämäräinen, Jari; Knoop, Henning; Stanford, Natalie J.; Guerrero, Fernando; Akhtar, M. Kalim; Aro, Eva‐Mari; Steuer, Ralf; Jones, Patrik R.

doi:10.1016/j.jbiotec.2012.07.193

Cited by 51 publications

(41 citation statements)

References 33 publications

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“…Growth rates were markedly reduced at concentrations higher than Bϩϩ. These results are consistent with a recent study showing that butanol is more toxic to Synechocystis than ethanol (30). Slower growth at these high n-butanol concentrations was accompanied by an increase in intracellular ROS levels (Fig.…”

Section: Resultssupporting

confidence: 91%

Using Transcriptomics To Improve Butanol Tolerance of Synechocystis sp. Strain PCC 6803

Anfelt

Hallström

Nielsen

et al. 2013

Appl Environ Microbiol

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c Cyanobacteria are emerging as promising hosts for production of advanced biofuels such as n-butanol and alkanes. However, cyanobacteria suffer from the same product inhibition problems as those that plague other microbial biofuel hosts. High concentrations of butanol severely reduce growth, and even small amounts can negatively affect metabolic processes. An understanding of how cyanobacteria are affected by their biofuel product can enable identification of engineering strategies for improving their tolerance. Here we used transcriptome sequencing (RNA-Seq) to assess the transcriptome response of Synechocystis sp. strain PCC 6803 to two concentrations of exogenous n-butanol. Approximately 80 transcripts were differentially expressed at 40 mg/liter butanol, and 280 transcripts were different at 1 g/liter butanol. Our results suggest a compromised cell membrane, impaired photosynthetic electron transport, and reduced biosynthesis. Accumulation of intracellular reactive oxygen species (ROS) scaled with butanol concentration. Using the physiology and transcriptomics data, we selected several genes for overexpression in an attempt to improve butanol tolerance. We found that overexpression of several proteins, notably, the small heat shock protein HspA, improved tolerance to butanol. Transcriptomics-guided engineering created more solventtolerant cyanobacteria strains that could be the foundation for a more productive biofuel host. C yanobacteria are attractive biochemical production platforms due to their minimal nutrient requirements, ease of genetic manipulation, and wide natural diversity. Several model cyanobacteria have been engineered as hosts for production of chemicals and biofuels such as hydrogen (1), ethanol (2), isobutanol (3, 4), and n-butanol (5). The modified cyanobacterial strains contained enzymes from fermentative organisms, such as Clostridium, Lactococcus, and Zymomonas species. The alcohols were produced at levels (up to 450 mg/liter) that were detectable but well below the titers of 10 to 20 g/liter achieved by industrial Clostridium and Saccharomyces strains (6). In addition to restrictions on metabolic flux (7,8), hosts may suffer from product inhibition even at low levels of product (9). Biofuel hosts that are more tolerant to the product may be more efficient producers even at levels below toxicity (10, 11), though this phenomenon is not universal (12).The deleterious effects of solvents on a wide range of microbes have been reported and recently reviewed (13). Solvents compromise the cell membrane and alter membrane fluidity (14). A compromised cell membrane can leak metabolites, resulting in loss of the transmembrane electrochemical gradient and hence of the proton-motive force. Reactive oxygen species (ROS) may accumulate as the cell modulates respiration to recover lost ATP (9, 15). These effects have been observed for bacteria, archaea, and yeast. Recent studies have begun to describe the complex response of cyanobacteria to solvents such as ethanol (16), n-butanol (17), and hexane (...

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Section: Resultssupporting

confidence: 91%

Using Transcriptomics To Improve Butanol Tolerance of Synechocystis sp. Strain PCC 6803

Anfelt

Hallström

Nielsen

et al. 2013

Appl Environ Microbiol

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“…Considering NADPH accumulation throughout the growth, it seems that efficient limonene production requires a lower ratio of ATP/NADPH. This observation is in agreement with a previous modeling study which revealed the lower ATP/NADPH ratio requirement for many biofuel molecules (28). In photosynthesis light reaction, linear electron flow is believed to produce ATP/NADPH in a ratio of approximately 1.28/1 (4).…”

Section: Photosynthesis Limitations In Enhancing Limonene Productionsupporting

confidence: 93%

“…Biomass accumulation is believed to require ATP/NADPH in a ratio of at least 1.51/1 (29), in which additional ATP is supplied by alternative electron flows (4). However, a nonnative carbon sink usually lacks the pathway complexity, and requires a smaller ATP/NADPH ratio compared with biomass accumulation (28). The photosynthesis light reaction thus might not require additional stimulation to accommodate the energy needs in these nonnative carbon sinks.…”

Section: Photosynthesis Limitations In Enhancing Limonene Productionmentioning

confidence: 99%

Enhanced limonene production in cyanobacteria reveals photosynthesis limitations

Wang

Liu

Xin

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

140

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Terpenes are the major secondary metabolites produced by plants, and have diverse industrial applications as pharmaceuticals, fragrance, solvents, and biofuels. Cyanobacteria are equipped with efficient carbon fixation mechanism, and are ideal cell factories to produce various fuel and chemical products. Past efforts to produce terpenes in photosynthetic organisms have gained only limited success. Here we engineered the cyanobacterium Synechococcus elongatus PCC 7942 to efficiently produce limonene through modeling guided study. Computational modeling of limonene flux in response to photosynthetic output has revealed the downstream terpene synthase as a key metabolic flux-controlling node in the MEP (2-C-methyl-D-erythritol 4-phosphate) pathway-derived terpene biosynthesis. By enhancing the downstream limonene carbon sink, we achieved over 100-fold increase in limonene productivity, in contrast to the marginal increase achieved through stepwise metabolic engineering. The establishment of a strong limonene flux revealed potential synergy between photosynthate output and terpene biosynthesis, leading to enhanced carbon flux into the MEP pathway. Moreover, we show that enhanced limonene flux would lead to NADPH accumulation, and slow down photosynthesis electron flow. Fine-tuning ATP/NADPH toward terpene biosynthesis could be a key parameter to adapt photosynthesis to support biofuel/ bioproduct production in cyanobacteria.photosynthesis | limonene | advanced biofuel | terpene | MEP E fficient carbon partition into desired molecules is a major scientific challenge in producing chemicals in photosynthetic organisms (1). Earlier approaches often involved overexpressing pathway enzymes to enhance carbon flux, but these approaches were hindered by the limited understanding of metabolic network and its regulation. In particular, many low-flux pathways (e.g., terpene biosynthesis) impede carbon partition due to metabolic rigidity (2, 3). Moreover, the importance and requirement of energy balance in improving photosynthetic productivity (4), is often neglected in engineering efforts. Recently, a few studies have demonstrated the possibility of producing terpenes in cyanobacteria, but the productivity is rather low (2, 5-7). Enhancing carbon flux into a low-flux terpene pathway could provide intuitive insight to both carbon partition and photosynthesis regulations. Through computational modeling, we show that downstream limonene synthase is a key flux-controlling node in the 2-C-methyl-Derythritol 4-phosphate (MEP)-derived limonene biosynthesis in cyanobacteria. Overcoming this metabolic bottleneck led to a record limonene productivity in the engineered cyanobacteria. Moreover, we show that enhanced limonene production led to redox change and energy imbalance, which ultimately limit photosynthesis capacity. The study demonstrates a successful strategy to enhance carbon partition into MEP-derived terpene biosynthesis, and reveals key photosynthesis regulations in providing ATP/NADPH to support terpene production. Stepwise M...

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“…In order to maximize the chance for novel renewable technologies to be commercialized, we argue that it is important to identify an optimal process and molecular target with respect to the entire chain from CO 2 to fuel and then eventually back to CO 2 again following combustion, including separation, chemical processing, storage and distribution there in between. The best molecular choice for all these considerations is not obvious and with few exceptions3536 a wide range of criteria have not been considered. For example, although butanol has higher energy density and lower water solubility compared with ethanol, it exhibits greater toxicity to biotechnological strains37.…”

Section: Discussionmentioning

confidence: 99%

“…For example, although butanol has higher energy density and lower water solubility compared with ethanol, it exhibits greater toxicity to biotechnological strains37. With the constraint of identifying a novel target that is well suited for the entire chain of production, distribution and utilization, propane was proposed for the following reasons: (i) alkanes are less toxic than their corresponding alcohols and acids36, (ii) the gaseous state of propane at common atmospheric conditions allows spontaneous separation during production, (iii) it can be stored in a high-density liquid state and (iv) it has excellent compatibility with the current fuel infrastructure for distribution and utilization (that is, liquefied petroleum gas).…”

Section: Discussionmentioning

confidence: 99%

An engineered pathway for the biosynthesis of renewable propane

et al. 2014

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The deployment of next-generation renewable biofuels can be enhanced by improving their compatibility with the current infrastructure for transportation, storage and utilization. Propane, the bulk component of liquid petroleum gas, is an appealing target as it already has a global market. In addition, it is a gas under standard conditions, but can easily be liquefied. This allows the fuel to immediately separate from the biocatalytic process after synthesis, yet does not preclude energy-dense storage as a liquid. Here we report, for the first time, a synthetic metabolic pathway for producing renewable propane. The pathway is based on a thioesterase specific for butyryl-acyl carrier protein (ACP), which allows native fatty acid biosynthesis of the Escherichia coli host to be redirected towards a synthetic alkane pathway. Propane biosynthesis is markedly stimulated by the introduction of an electron-donating module, optimizing the balance of O 2 supply and removal of native aldehyde reductases.

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Physiological tolerance and stoichiometric potential of cyanobacteria for hydrocarbon fuel production

Cited by 51 publications

References 33 publications

Using Transcriptomics To Improve Butanol Tolerance of Synechocystis sp. Strain PCC 6803

Using Transcriptomics To Improve Butanol Tolerance of Synechocystis sp. Strain PCC 6803

Enhanced limonene production in cyanobacteria reveals photosynthesis limitations

An engineered pathway for the biosynthesis of renewable propane

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